Bioluminescence eliminates need for excitation source
Hank Hogan, firstname.lastname@example.org
Quantum dots are useful for biological imaging because they are stable
and bright, and their emission depends on their size. Researchers can pick quantum
dots of a certain emission to match their application or use several sizes for multiplexing.
They may soon have a new choice: self-illuminating quantum dots.
Scientists from Stanford University in California
recently developed a way to modify quantum dots so that they provide their own excitation
light by converting chemical energy into light. Because no excitation light is necessary,
the dots virtually eliminate autofluorescence and could provide a way to image deep
within the tissue of living animals.
Typically, quantum dots consist of
a heavy metal or semiconductor core, such as cadmium selenide. Because the core
is toxic, it’s sealed, often with a zinc-sulfide shell covered by an organic
coating. The use of a shell also has the advantage of rendering the quantum dot
water-soluble. The core-shell structure, a few tens of nanometers in diameter, emits
in the visible when excited by an external light source of the right wavelength.
There are some downsides to quantum dots, including
questions about their safety and size. Both are problems for in vivo applications.
However, one of the biggest disadvantages is one that the researchers experienced
firsthand. “We began to work on quantum dot in vivo fluorescence imaging first
and ran into the problem of autofluorescence,” said Jianghong Rao of Stanford’s
School of Medicine.
Self-illuminating quantum dots were used to image C6 glioma cells in vitro and in mice. On the left
is a bioluminescence image of a nude mouse injected via the tail with the labeled
cells, which accumulated from deep lung tissue. On the right is an overlay of fluorescence
and differential interference contrast images of cells labeled with self-illuminating
quantum dots, conjugates of a quantum dot that emits at 655 nm and Luc8 luciferase.
Courtesy of Jianghong Rao and Stanford University.
The external light source used to get
quantum dot emission is the same wavelength that excites fluorescence in a host
of organic compounds found in abundance in living animal cells. The list includes
collagens, porphyrins and flavins. In addition, because tissue scatters and absorbs
the excitation light before it gets to the quantum dots, the signal from the dot
is weakened and must compete with the autofluorescence of the surrounding tissue.
At the same time the researchers were
attempting quantum dots fluorescence imaging, they also were working on in vivo
bioluminescence imaging. The contrast between results from the two methods was stark.
While the quantum dots signal was drowning in a flood of unwanted light, the bioluminescent
imaging showed high sensitivity and low background noise.
Therefore, they began to work on ways
to bioluminescently image quantum dots. The key to achieving this involved the creation
of a light source that was part of the probe itself.
For this, they turned to bioluminescence
resonance energy transfer (BRET), a naturally occurring phenomenon in which a light-emitting
protein, the donor, transfers energy to a fluorescent protein, the acceptor, when
the two are close together. BRET is similar to Förster resonance energy transfer
(FRET); however, unlike FRET, the donor’s energy comes from a chemical reaction.
FRET is known to occur between quantum dot donors and organic dye acceptors, but
it wasn’t known whether the dots could act as a BRET acceptor.
To find out, the investigators selected
luciferase from the sea pansy Renilla reniformis because its 480-nm emission
peak was bluer than that of other luciferases, an important consideration because
quantum dots absorb bluer light more efficiently. This luciferase is typically used
in bioluminescence imaging and, like luciferases found in fireflies and elsewhere,
it emits light in the presence of oxygen and a substrate, luciferin. In this case,
the luciferin was coelenterazine.
Through an extensive process of mutagenesis,
they created an eight-mutation variant designated Luc8 that was more stable in serum
and had improved catalytic efficiency than the wild-type protein. They attached
the variant to CdSe/ZnS quantum dots (QD655) from Quantum Dot Corp. of Hayward,
Calif., that had a fluorescence emission at 655 nm. “The conjugation of the
protein to quantum dots is rather straightforward, a simple amide formation chemistry,
but it took a lot of time to optimize the procedures to avoid aggregations of the
product,” Rao said.
The layer of luciferase increased the
size of the quantum dots by only 2 nm, with the diameter of the conjugate at 22
nm. In the presence of a sufficient concentration of coelenterazine, the luciferase
emitted light and initiated BRET with the quantum dots. This produced 655-nm fluorescence
from the acceptors without an external light source. Thus, there was also minimal
After verifying that the QD655-Luc8
conjugate worked in mouse serum and whole blood, the researchers injected it into
mice about 3 mm below the skin, in the muscles. They injected coelenterazine and
imaged the mouse, with and without a filter. They used a bioluminescence imaging
system from Xenogen Corp. of Alameda, Calif., with a 30-s image acquisition time
and found that they could detect the conjugate from millimeters deep within the
animal. On the other hand, they spotted the luciferase emission only at the surface.
Using quantum dots of different sizes as the BRET
acceptor, they multiplexed the imaging of conjugates at emission peaks of 605, 655,
705 and 800 nm. They were able to unmix the various spectral components, employing
an imaging system and specialized software from CRI Inc. of Woburn, Mass. The work
was published in the March issue of Nature Biotechnology.
The technique could be useful for applications
that are difficult for standard quantum dots.
“The light emission from the
quantum dots can be turned on and off by a biological interaction,” Rao said.
That capability could be used to build nanosensors that emit light in response to
specific biological activity.
Rao readily acknowledged that this
approach doesn’t get around any potential problems due to quantum dot size
or toxicity, adding that a small and nontoxic probe would be best for in vivo imaging.
He noted that ongoing research efforts aim to resolve these issues.
However, the researchers are not waiting
for such innovations before attempting to realize the potential of quantum dots
conjugates for in vivo imaging. “The next step we are taking is to apply it
to cancer imaging by attaching tumor cell-specific molecules to the quantum dots,
and to image multiple tumor targets at the same time with multiplexed imaging,”
He also noted that the technique is
not limited to a particular luciferase. It could potentially be used with other